Transgenic brassica event MON 88302 and methods of use thereof

ABSTRACT

The invention provides plants comprising transgenic event MON 88302 that exhibit tolerance to glyphosate herbicide. The invention also provides seeds, plant parts, cells, commodity products, and methods related to the event. The invention also provides DNA molecules that are unique to the event and were created by the insertion of transgenic DNA into the genome of a  Brassica napus  plant.

This application is a divisional of co-pending U.S. patent applicationSer. No. 13/151,082, filed Jun. 1, 2011, which claims the benefit ofU.S. Provisional Application No. 61/351,317, filed on Jun. 4, 2010, theentire disclosure of which is incorporated herein by reference.

INCORPORATION OF SEQUENCE LISTING

The sequence listing that is contained in the file named“38-21_56992_seqlisting_ST25.txt”, which is 20.7 kilobytes (size asmeasured in Microsoft Windows®) and was created on Jun. 2, 2010, isfiled herewith by electronic submission and is incorporated by referenceherein.

FIELD OF THE INVENTION

The invention relates to the fields of biotechnology and agriculture andmore specifically to the field of transgenic crop plants.

BACKGROUND OF THE INVENTION

Brassica crops are important in many areas of the world. The methods ofbiotechnology may be applied to these crops to produce crops withimproved traits such as herbicide tolerance. Herbicide tolerance may beachieved in transgenic plants by the expression of a transgene capableof providing such tolerance. The expression of a transgene in a plantmay be influenced by a combination of factors such as the regulatoryelements used in the transgene cassette, the chromosomal location of thetransgene insert, and the proximity of any endogenous regulatoryelements close to the integration site. For example, it has beenobserved that there may be wide variation in the overall level oftransgene expression or in the spatial or temporal pattern of transgeneexpression between similarly-produced events. For this reason, it may benecessary to produce and test hundreds of individual planttransformation events in order to ultimately identify one event usefulfor commercial agricultural purposes. Such an event, once identified ashaving the desired transgene expression and molecular characteristics,may then be used for introgressing the trait into other geneticbackgrounds using plant breeding methods. The resulting progeny wouldcontain the transgenic event and would therefore have the transgeneexpression characteristics for that trait of the original transformant.This may be used to produce a number of different crop varieties thatcomprise the improved trait and are suitably adapted to specific localgrowing conditions.

SUMMARY OF THE INVENTION

The invention provides transgenic plants and seeds comprising event MON88302, a representative seed sample of which has been deposited withAmerican Type Culture Collection (ATCC) with Accession No. PTA-10955.Plants comprising the event exhibit commercially acceptable tolerance toapplications of glyphosate herbicide. The invention provides progenyplants, plant parts, and cells comprising the event; recombinant DNAmolecules related to the event and methods of using these molecules;commodity products derived from or comprising the event; and methods ofusing the event.

The invention provides a plant, seed, cell, progeny plant, or plant partcomprising the event and commodity products derived from a plant, cell,plant part, or seed comprising the event. The invention thus provides aplant, seed, cell, progeny plant, plant part, or commodity productcomprising a DNA molecule having a nucleotide sequence selected from thegroup consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO:4, SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8, and fragments thereof.The invention provides a plant, seed, cell, progeny plant, or plant partcomprising a recombinant DNA molecule that produces an ampliconcomprising a DNA molecule of the invention, for instance in a DNAamplification method.

The invention provides DNA molecules related to the event. These DNAmolecules may comprise nucleotide sequences representing or derived fromthe junction of the transgene insertion and flanking genomic DNA ofevent MON 88302, and/or a region of the genomic DNA flanking theinserted DNA, and/or a region of the integrated transgenic DNA flankingthe insertion site, and/or a region of the integrated transgenicexpression cassette, and/or a contiguous sequence of any of theseregions. The invention also provides DNA molecules useful as primers andprobes diagnostic for the event. Plants, cells, plant parts, commodityproducts, progeny, and seeds comprising these molecules are provided.

The invention provides methods, compositions, and kits useful fordetecting the presence of DNA derived from the event. The inventionprovides a method for detection of the event by contacting a samplecomprising DNA with a primer set that when used in a nucleic acidamplification reaction with genomic DNA from the event produces anamplicon diagnostic for the event, performing a nucleic acidamplification reaction thereby producing the amplicon, and detecting theamplicon. The invention also provides a method for detection of theevent by contacting a sample comprising DNA with a probe that when usedin a hybridization reaction with genomic DNA from the event hybridizesto a DNA molecule specific for the event, performing a hybridizationreaction, and detecting the hybridization of the probe to the DNAmolecule. Kits comprising the methods and compositions of the inventionuseful for detecting the presence of DNA derived from the event are alsoprovided.

The invention provides a method for controlling weeds in a field byplanting plants comprising the event (i.e., planting seeds comprisingthe events) and then applying an effective dose of glyphosate capable ofcontrolling the weeds without injuring the plants comprising the event.

The invention provides methods of producing a plant and/or seed thattolerates application of glyphosate herbicide by crossing a glyphosatetolerant plant comprising the event or comprising a sequence selectedfrom the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3,SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8 with a secondplant, thereby producing seed, growing the seed to produce progenyplants, treating the progeny plants with glyphosate, and selecting aprogeny plant that comprises the event and is tolerant to glyphosate.The invention provides methods of producing a plant and/or seed thattolerates application of glyphosate herbicide by selfing a glyphosatetolerant plant comprising the event or comprising a sequence selectedfrom the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3,SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8, therebyproducing seed, growing the seed to produce progeny plants, treating theprogeny plants with glyphosate; and selecting a progeny plant thatcomprises the event and is tolerant to glyphosate.

The invention provides methods of determining the zygosity of a plant orseed comprising the event, by contacting a sample comprising DNA with afirst primer set that when used in a nucleic acid amplification reactionwith genomic DNA from event MON 88302 produces an amplicon diagnosticfor the event, performing a nucleic acid amplification reaction therebyproducing the amplicon, detecting the amplicon, contacting the samplewith a second primer set that when used in a nucleic-acid amplificationreaction with genomic DNA from plants produces a second ampliconcomprising the native genomic DNA homologous to the genomic region of atransgene insertion identified as event MON 88302, performing a nucleicacid amplification reaction thereby producing the second amplicon,detecting the second amplicon, and comparing the first and secondamplicons in a sample, wherein the presence of both amplicons indicatesthe sample and thus the plant or seed is heterozygous for the transgeneinsertion.

The foregoing and other aspects of the invention will become moreapparent from the following detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Diagrammatical representation of event MON 88302; [A]corresponds to the relative position of the 5′ junction region; [B]corresponds to the relative position of the 3′ junction region; [C]corresponds to the relative position of the flanking region and aportion of the 5′ end of the inserted transgenic DNA; [D] corresponds tothe relative position of the 3′ flanking region and a portion of the 3′end of the inserted transgenic DNA; [E] represents the transgeneexpression cassette; and [F] represents the contiguous sequence of theBrassica napus genomic flanking sequences and transgene expressioncassette.

FIG. 2: Shows grain yield of RT73 event compared to MON 88302 whenglyphosate is applied at the four to six leaf stage. RT73 is designatedas RR1.

FIG. 3: Shows grain yield of RT73 event compared to MON 88302 whenglyphosate is applied at first flower. RT73 is designated as RR1.

BRIEF DESCRIPTION OF THE SEQUENCES

SEQ ID NO: 1—A sixty nucleotide sequence representing the 5′ junctionsequence between the Brassica napus genomic DNA and the integratedtransgenic expression cassette. This nucleotide sequence corresponds topositions 762 through 821 of SEQ ID NO: 3 ([C], see FIG. 1) and topositions 762 through 821 of SEQ ID NO: 6.

SEQ ID NO: 2—A sixty nucleotide sequence representing the 3′ junctionbetween the integrated expression cassette and the Brassica napusgenomic DNA. This nucleotide sequence corresponds to positions 313through 372 of SEQ ID NO: 4 ([D], see FIG. 1), and to positions 5189through 5248 of SEQ ID NO: 6.

SEQ ID NO: 3—The 5′ sequence flanking the inserted DNA of event MON88302 up to and including a region of transgenic DNA. Nucleotidepositions 762 through 821 of SEQ ID NO: 3 correspond to nucleotidepositions 1 through 60 of SEQ ID NO: 1; nucleotide positions 742 through841 of SEQ ID NO: 3 correspond to nucleotide positions 1 through 100 ofSEQ ID NO: 7 and nucleotide positions 792 through 956 of SEQ ID NO: 3correspond to nucleotide positions 1 through 165 of SEQ ID NO: 5.

SEQ ID NO: 4—The 3′ sequence flanking the inserted DNA of event MON88302 up to and including a region of transgenic DNA. Nucleotidepositions 313 through 372 of SEQ ID NO: 4 correspond to nucleotidepositions 1 through 60 of SEQ ID NO: 2; nucleotide positions 293 through392 of SEQ ID NO: 4 correspond to nucleotide positions 1 through 100 ofSEQ ID NO: 8 and the nucleotide positions 1 through 342 of SEQ ID NO: 4correspond to nucleotide positions 4086 through 4427 of SEQ ID NO: 5.

SEQ ID NO: 5—The sequence of the integrated transgenic expressioncassette conferring glyphosate herbicide tolerance. SEQ ID NO: 5corresponds to nucleotide positions 792 through 5218 of SEQ ID NO: 6.

SEQ ID NO: 6—A nucleotide sequence representing the contig of the 5′sequence flanking the inserted DNA of event MON 88302 (SEQ ID NO: 3),the sequence of the integrated expression cassette (SEQ ID NO: 5), andthe 3′ sequence flanking the inserted DNA of event MON 88302 (SEQ ID NO:4).

SEQ ID NO: 7—A 100-nucleotide sequence representing the 5′ junctionsequence between the Brassica napus genomic DNA and the integratedtransgenic expression cassette. This nucleotide sequence corresponds topositions 742 through 841 of SEQ ID NO: 3 ([C], see FIG. 1) and toposition 742 through 841 of SEQ ID NO: 6.

SEQ ID NO: 8—A 100-nucleotide sequence representing the 3′ junctionbetween the integrated expression cassette and the Brassica napusgenomic DNA. This nucleotide sequence corresponds to positions 293through 392 of SEQ ID NO: 4 ([D], see FIG. 1), and to positions 5169through 5268 of SEQ ID NO: 6.

SEQ ID NO: 9—Primer SQ20901 used to identify event MON 88302. PrimerSQ20901 is complementary to the inserted expression cassette at theregion close to the 3′ transgene insertion border. An amplicon producedusing the combination of primers SQ20901 and SQ23770 (SEQ ID NO: 10) isa positive result for the presence of the event MON 88302.

SEQ ID NO: 10 is the sequence of a primer referred to as Primer SQ23770and used to identify event MON 88302. Primer SQ23770 is complimentary toa 3′ region flanking the inserted expression cassette and close to thetransgene DNA insertion border. An amplicon produced using thecombination of primers SQ20901 (SEQ ID NO: 9) and SQ23770 is a positiveresult for the presence of the event MON 88302.

SEQ ID NO: 11 is the sequence of a probe referred to as Probe PB 10164and used to identify event MON 88302. It is complimentary to a 3′ regionflanking the inserted expression cassette and close to the transgene DNAinsertion border. This probe is a 6FAM™-labeled syntheticoligonucleotide. Release of a fluorescent signal in an amplificationreaction using primers SQ20901 and SQ23770 (SEQ ID NOs: 9-10) incombination with 6FAM™-labeled probe PB10164 is diagnostic of event MON88302 in a TAQMAN® assay.

SEQ ID NO: 12 is the sequence of a primer referred to as Primer SQ21948and used to identify MON 88302 event zygosity.

SEQ ID NO: 13 is the sequence of a primer referred to as Primer SQ24635and used to identify Brassica napus wild-type zygosity.

SEQ ID NO: 14 is the sequence of a primer referred to as Primer SQ22176and used to identify MON 88302 event and Brassica napus wild-typezygosity.

SEQ ID NO: 15 is the sequence of a probe (PB4213) for a MON 88302 eventzygosity assay.

SEQ ID NO: 16 is the sequence of a probe (PB10787) for a Brassica napuswild-type zygosity assay.

SEQ ID NO: 17 is the sequence of a primer referred to as Primer SQ2563and used as an internal control in the TAQMAN® assays.

SEQ ID NO: 18 is the sequence of a primer referred to as Primer SQ2564and used as an internal control in the TAQMAN® assays.

SEQ ID NO: 19 is the sequence of a VIC™-labeled syntheticoligonucleotide probe (PB0751) used as an internal control in theTAQMAN® assays.

DETAILED DESCRIPTION

The following definitions and methods are provided to better define thepresent invention and to guide those of ordinary skill in the art in thepractice of the present invention. Unless otherwise noted, terms are tobe understood according to conventional usage by those of ordinary skillin the relevant art.

As used herein, the term “comprising” means “including but not limitedto”.

The present invention provides transgenic event MON 88302. The term“event” as used herein refers to DNA molecules produced as a result ofinserting transgenic DNA into a plant's genome at a particular locationon a chromosome. Event MON 88302 refers to the DNA molecules produced asa result of the insertion of transgenic DNA having a sequence providedherein as SEQ ID NO: 5 into a particular location in the Brassica napusA genome on linkage group N4. Plants and seeds comprising event MON88302 are also provided in the present invention. A seed samplecontaining MON 88302 has been deposited with American Type CultureCollection (ATCC) with Accession No. PTA-10955. Plants comprising MON88302 exhibit commercially acceptable tolerance to applications ofglyphosate herbicide.

A plant comprising the event can refer to the original transformant thatincludes the transgene inserted into the particular location in theplant's genome. A plant comprising the event can also refer to progenyof the original transformant that include the transgene inserted intothe particular location in the plant's genome. Such progeny may beproduced by selfing or by a sexual outcross between the transformant, orits progeny, and another plant. Such other plant may be a transgenicplant comprising the same or different transgene and/or a nontransgenicplant, such as one from a different variety. Even after repeatedback-crossing to a recurrent parent, the inserted DNA and flanking DNAfrom the transformed parent is present in the progeny of the cross atthe same genomic location.

Transgenic event MON 88302 was created by the insertion of transgenicDNA (provided herein as SEQ ID NO: 5) into linkage group N4 of the Agenome of a Brassica napus plant. Brassica napus is commonly known asrapeseed and specific cultivars may be referred to as canola. As usedherein, the term “canola” or “canola plant” refers to a Brassica plantcapable of being used to produce canola oil (i.e. oil meeting a specificquality designation of containing less than 2% erucic acid) and includesvarieties of Brassica napus, Brassica napobrassica, Brassica rapa,Brassica juncea, and Brassica campestris. Because Brassica napus is anallotetraploid arising from the cross and retention of both genomes ofBrassica rapa (previously Brassica campestris) and Brassica oleracea, aBrassica napus plant comprising transgenic event MON 88302 may be usedwith breeding methods to introduce the MON 88302 event, and thus theglyphosate tolerance trait, into other members of the Brassica genus.Examples of members of the Brassica genus useful in practicing themethods of the invention include but are not limited to Brassica juncea,Brassica napobrassica, Brassica oleracea, Brassica carinata, Brassicanapus, Brassica rapa, and Brassica campestris, as well as any otherplants belonging to the genus Brassica that permit breeding betweenBrassica species.

A DNA molecule comprising event MON 88302 refers to a DNA moleculecomprising at least a portion of the inserted transgenic DNA (providedas SEQ ID NO: 5) and at least a portion of the flanking genomic DNAimmediately adjacent to the inserted DNA. As such, a DNA moleculecomprising event MON 88302 has a nucleotide sequence representing atleast a portion of the transgenic DNA insert and at least a portion ofthe particular region of the genome of the plant into which thetransgenic DNA was inserted. The arrangement of the inserted DNA inevent MON 88302 in relation to the surrounding plant genome is specificand unique to event MON 88302 and as such the nucleotide sequence ofsuch a DNA molecule is descriptive and identifying for event MON 88302.Examples of the sequence of such a DNA molecule are provided herein asSEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 6,SEQ ID NO: 7, and SEQ ID NO: 8. This DNA molecule is also an integralpart of the chromosome of a plant that comprises event MON 88302 and maybe passed on to progeny of the plant.

Event MON 88302 confers tolerance to glyphosate herbicide applied to theplant. “Glyphosate” refers to N-phosphonomethyl-glycine and its salts.Glyphosate is an herbicide that has activity on a broad spectrum ofplant species. When applied to a plant surface, glyphosate movessystemically through the plant. Glyphosate is phototoxic due to itsinhibition of the shikimic acid pathway, which provides a precursor forthe synthesis of aromatic amino acids.

As used herein, the term “recombinant” refers to a form of DNA and/orprotein and/or an organism that would not normally be found in natureand as such was created by human intervention. Such human interventionmay produce a recombinant DNA molecule and/or a recombinant plant. Asused herein, a “recombinant DNA molecule” is a DNA molecule comprising acombination of DNA molecules that would not naturally occur together andis the result of human intervention, e.g., a DNA molecule that iscomprised of a combination of at least two DNA molecules heterologous toeach other, and/or a DNA molecule that is artificially synthesized andcomprises a polynucleotide sequence that deviates from thepolynucleotide sequence that would normally exist in nature, and/or aDNA molecule that comprises a transgene artificially incorporated into ahost cell's genomic DNA and the associated flanking DNA of the hostcell's genome. An example of a recombinant DNA molecule is a DNAmolecule described herein resulting from the insertion of the transgeneinto the Brassica napus genome, which may ultimately result in theexpression of a recombinant RNA and/or protein molecule in thatorganism. As used herein, a “recombinant plant” is a plant that wouldnot normally exist in nature, is the result of human intervention, andcontains a transgene and/or heterologous DNA molecule incorporated intoits genome. As a result of such genomic alteration, the recombinantplant is distinctly different from the related wildtype plant. Anexample of a recombinant plant is a plant described herein as comprisingevent MON 88302.

As used herein, the term “transgene” refers to a polynucleotide moleculeartificially incorporated into a host cell's genome. Such transgene maybe heterologous to the host cell. The term “transgenic plant” refers toa plant comprising such a transgene.

As used herein, the term “heterologous” refers to a first molecule notnormally found in combination with a second molecule in nature. Forexample, a molecule may be derived from a first species and insertedinto the genome of a second species. The molecule would thus beheterologous to the host and artificially incorporated into a hostcell's genome.

As used herein, the term “chimeric” refers to a single DNA moleculeproduced by fusing a first DNA molecule to a second DNA molecule, whereneither first nor second DNA molecule would normally be found in thatconfiguration, i.e., fused to the other. The chimeric DNA molecule isthus a new DNA molecule not otherwise normally found in nature.

The present invention provides DNA molecules and their correspondingnucleotide sequences. As used herein, the terms “DNA sequence”,“nucleotide sequence” and “polynucleotide sequence” refer to thesequence of nucleotides of a DNA molecule, usually presented from the 5′(upstream) end to the 3′ (downstream) end. The nomenclature used hereinis that required by Title 37 of the United States Code of FederalRegulations §1.822 and set forth in the tables in WIPO Standard ST.25(1998), Appendix 2, Tables 1 and 3. The present invention is disclosedwith reference to only one strand of the two nucleotide sequence strandsthat are provided in transgenic event MON 88302. Therefore, byimplication and derivation, the complementary sequences, also referredto in the art as the complete complement or the reverse complementarysequences, are within the scope of the present invention and aretherefore also intended to be within the scope of the subject matterclaimed.

The nucleotide sequence corresponding to the complete nucleotidesequence of the inserted transgenic DNA and substantial segments of theBrassica napus genomic DNA flanking either end of the insertedtransgenic DNA is provided herein as SEQ ID NO: 6. A subsection of thisis the inserted transgenic DNA provided as SEQ ID NO: 5. The nucleotidesequence of the genomic DNA flanking the 5′ end of the insertedtransgenic DNA and a portion of the 5′ end of the inserted DNA isprovided herein as SEQ ID NO: 3. The nucleotide sequence of the genomicDNA flanking the 3′ end of the inserted transgenic DNA and a portion ofthe 3′ end of the inserted DNA is provided herein as SEQ ID NO: 4. Theregion spanning the location where the transgenic DNA connects to and islinked to the genomic DNA, is referred to herein as the junction. A“junction sequence” or “junction region” refers to a DNA sequence and/orcorresponding DNA molecule that spans the inserted transgenic DNA andthe adjacent flanking genomic DNA. Examples of a junction sequence ofevent MON 88302 are provided herein as SEQ ID NO: 1, SEQ ID NO: 2, SEQID NO: 7, and SEQ ID NO: 8. The identification of one of these junctionsequences in a nucleotide molecule derived from a Brassica plant or seedis conclusive that the DNA was obtained from event MON 88302 and isdiagnostic for the presence in a sample of DNA from event MON 88302. SEQID NO: 1 is a 60 nucleotide sequence spanning the junction between thegenomic DNA and the 5′ end of the inserted DNA. SEQ ID NO: 7 is a 100nucleotide sequence spanning the junction between the genomic DNA andthe 5′ end of the inserted DNA. SEQ ID NO: 2 is a 60 nucleotide sequencespanning the junction between the genomic DNA and the 3′ end of theinserted DNA. SEQ ID NO: 8 is a 100 nucleotide sequence spanning thejunction between the genomic DNA and the 3′ end of the inserted DNA. Anysegment of DNA derived from transgenic event MON 88302 that includes SEQID NO: 1 or SEQ ID NO: 7 is within the scope of the present invention.Any segment of DNA derived from transgenic event MON 88302 that includesSEQ ID NO: 2 or SEQ ID NO: 8 is within the scope of the presentinvention. In addition, any polynucleotide comprising a sequencecomplementary to any of the sequences described within this paragraph iswithin the scope of the present invention. FIG. 1 illustrates thephysical arrangement of SEQ ID NOs: 1-5 and SEQ ID NOs: 7-8 relative toSEQ ID NO: 6 arranged from 5′ to 3′. The present invention also providesa nucleic acid molecule comprising a DNA molecule having a sequence thatis at least 95%, 96%, 97%, 98%, or 99% identical to the full-length ofSEQ ID NO: 6.

The present invention provides exemplary DNA molecules that can be usedeither as primers or probes for diagnosing the presence of DNA derivedfrom event MON 88302 in a sample. Such primers or probes are specificfor a target nucleic acid sequence and as such are useful for theidentification of event MON 88302 nucleic acid sequence by the methodsof the invention described herein.

A “primer” is typically a highly purified, isolated polynucleotide thatis designed for use in specific annealing or hybridization methods thatinvolve thermal amplification. A pair of primers may be used withtemplate DNA, such as a sample of Brassica napus genomic DNA, in athermal amplification, such as polymerase chain reaction (PCR), toproduce an amplicon, where the amplicon produced from such reactionwould have a DNA sequence corresponding to sequence of the template DNAlocated between the two sites where the primers hybridized to thetemplate. As used herein, an “amplicon” is a piece or fragment of DNAthat has been synthesized using amplification techniques, i.e. theproduct of an amplification reaction. In one embodiment of theinvention, an amplicon diagnostic for event MON 88302 comprises asequence not naturally found in the Brassica napus genome. An ampliconof the present invention comprises at least about 40 contiguousnucleotides of SEQ ID NO: 1 or SEQ ID NO: 2, and complements thereof. Aprimer is typically designed to hybridize to a complementary target DNAstrand to form a hybrid between the primer and the target DNA strand,and the presence of the primer is a point of recognition by a polymeraseto begin extension of the primer (i.e., polymerization of additionalnucleotides into a lengthening polynucleotide molecule) using as atemplate the target DNA strand. Primer pairs, as used in the presentinvention, are intended to refer to use of two primers binding oppositestrands of a double stranded nucleotide segment for the purpose ofamplifying linearly the polynucleotide segment between the positionstargeted for binding by the individual members of the primer pair,typically in a thermal amplification reaction or other conventionalnucleic-acid amplification methods. Exemplary DNA molecules useful asprimers are provided as SEQ ID NOs: 9-10. The primer pair provided asSEQ ID NO: 9 and SEQ ID NO: 10 may be used as a first DNA molecule and asecond DNA molecule that is different from the first DNA molecule, andboth molecules are each of sufficient length of contiguous nucleotidesof either SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 6 or the complementsthereof to function as DNA primers so that, when used together in athermal amplification reaction with template DNA derived from event MON88302, an amplicon that is specific and unique to the 3′ portion oftransgenic event MON 88302 would be produced. This exemplary primer pairmay be used to amplify a 3′ junction region diagnostic for event MON88302. Similarly, the invention provides primer pairs that may be usedto amplify a 5′ junction region diagnostic for event MON 88302. Suchprimers may comprise a first DNA molecule and a second DNA molecule thatis different from the first DNA molecule, both of sufficient length ofcontiguous nucleotides of SEQ ID NO: 3, SEQ ID NO: 5, or SEQ ID NO: 6 orthe complements thereof to function as DNA primers so that, when usedtogether in a thermal amplification reaction with template DNA derivedfrom event MON 88302, an amplicon that is specific and unique to the 5′portion of transgenic event MON 88302 would be produced.

A “probe” is an isolated nucleic acid that is complementary to a strandof a target nucleic acid. Probes according to the present inventioninclude not only deoxyribonucleic or ribonucleic acids but alsopolyamides and other probe materials that bind specifically to a targetDNA sequence and the detection of such binding can be useful indiagnosing, discriminating, determining, or confirming the presence ofthat target DNA sequence in a particular sample. A probe may be attachedto a conventional detectable label or reporter molecule, e.g., aradioactive isotope, ligand, chemiluminescent agent, or enzyme. In oneembodiment of the invention, a probe diagnostic for event MON 88302comprises a sequence not naturally found in the Brassica napus genome.An exemplary DNA molecule useful as a probe is provided as SEQ ID NO:11.

Probes and primers according to the present invention may have completesequence identity with the target sequence, although primers and probesdiffering from the target sequence that retain the ability to hybridizepreferentially to target sequences may be designed by conventionalmethods. In order for a nucleic acid molecule to serve as a primer orprobe it need only be sufficiently complementary in sequence to be ableto form a stable double-stranded structure under the particular solventand salt concentrations employed. Any conventional nucleic acidhybridization or amplification method can be used to identify thepresence of transgenic DNA from event MON 88302 in a sample. Probes andprimers are generally at least about 11 nucleotides, at least about 18nucleotides, at least about 24 nucleotides, or at least about 30nucleotides or more in length. Such probes and primers hybridizespecifically to a target DNA sequence under stringent hybridizationconditions. Conventional stringency conditions are described by Sambrooket al., 1989, and by Haymes et al., In: Nucleic Acid Hybridization, APractical Approach, IRL Press, Washington, D.C. (1985). As used herein,two nucleic acid molecules are said to be capable of specificallyhybridizing to one another if the two molecules are capable of formingan anti-parallel, double-stranded nucleic acid structure. A nucleic acidmolecule is said to be the “complement” of another nucleic acid moleculeif they exhibit complete complementarity. As used herein, molecules aresaid to exhibit “complete complementarity” when every nucleotide of oneof the molecules is complementary to a nucleotide of the other. Twomolecules are said to be “minimally complementary” if they can hybridizeto one another with sufficient stability to permit them to remainannealed to one another under at least conventional “low-stringency”conditions. Similarly, the molecules are said to be “complementary” ifthey can hybridize to one another with sufficient stability to permitthem to remain annealed to one another under conventional“high-stringency” conditions. Departures from complete complementarityare therefore permissible, as long as such departures do not completelypreclude the capacity of the molecules to form a double-strandedstructure.

As used herein, the term “isolated” refers to at least partiallyseparating a molecule from other molecules normally associated with itin its native or natural state. In one embodiment, the term “isolated”refers to a DNA molecule that is at least partially separated from thenucleic acids which normally flank the DNA molecule in its native ornatural state. Thus, DNA molecules fused to regulatory or codingsequences with which they are not normally associated, for example asthe result of recombinant techniques, are considered isolated herein.Such molecules are considered isolated even when integrated into thechromosome of a host cell or present in a nucleic acid solution withother DNA molecules.

Any number of methods well known to those skilled in the art can be usedto isolate and manipulate a DNA molecule, or fragment thereof, disclosedin the present invention. For example, PCR (polymerase chain reaction)technology can be used to amplify a particular starting DNA moleculeand/or to produce variants of the original molecule. DNA molecules, or afragment thereof, can also be obtained by other techniques such as bydirectly synthesizing the fragment by chemical means, as is commonlypracticed by using an automated oligonucleotide synthesizer.

The DNA molecules and corresponding nucleotide sequences provided hereinare therefore useful for, among other things, identifying event MON88302, selecting plant varieties or hybrids comprising event MON 88302,detecting the presence of DNA derived from event MON 88302 in a sample,and monitoring samples for the presence and/or absence of event MON88302 or plants and plant parts comprising event MON 88302.

The present invention provides plants, progeny, seeds, plant cells,plant parts (such as pollen, ovule, pod, flower, root or stem tissue,fibers, and leaves), and commodity products. These plants, progeny,seeds, plant cells, plant parts, and commodity products contain adetectable amount of a polynucleotide of the present invention, i.e.,such as a polynucleotide having at least one of the sequences providedas SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 7, or SEQ ID NO: 8. Plants,progeny, seeds, plant cells, and plant parts of the present inventionmay also contain one or more additional transgenes. Such transgene maybe any nucleotide sequence encoding a protein or RNA molecule conferringa desirable trait including but not limited to increased insectresistance, increased water use efficiency, increased yield performance,increased drought resistance, increased seed quality, diseaseresistance, improved nutritional quality, and/or increased herbicidetolerance, in which the desirable trait is measured with respect to acomparable plant lacking such additional transgene.

The present invention provides plants, progeny, seeds, plant cells, andplant part such as pollen, ovule, pod, flower, root or stem tissue, andleaves derived from a transgenic plant comprising event MON 88302. Arepresentative sample of seed comprising event MON 88302 has beendeposited according to the Budapest Treaty for the purpose of enablingthe present invention. The repository selected for receiving the depositis the American Type Culture Collection (ATCC) having an address at10801 University Boulevard, Manassas, Va. USA, Zip Code 20110. The ATCCrepository has assigned the accession No. PTA-10955 to the event MON88302 seed.

The present invention provides a microorganism comprising a DNA moleculehaving SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 7, or SEQ ID NO: 8 presentin its genome. An example of such a microorganism is a transgenic plantcell. Microorganisms, such as a plant cell of the present invention, areuseful in many industrial applications, including but not limited to:(i) use as research tool for scientific inquiry or industrial research;(ii) use in culture for producing endogenous or recombinantcarbohydrate, lipid, nucleic acid, or protein products or smallmolecules that may be used for subsequent scientific research or asindustrial products; and (iii) use with modern plant tissue culturetechniques to produce transgenic plants or plant tissue cultures thatmay then be used for agricultural research or production. The productionand use of microorganisms such as transgenic plant cells utilizes modernmicrobiological techniques and human intervention to produce a man-made,unique microorganism. In this process, recombinant DNA is inserted intoa plant cell's genome to create a transgenic plant cell that is separateand unique from naturally occurring plant cells. This transgenic plantcell can then be cultured much like bacteria and yeast cells usingmodern microbiology techniques and may exist in an undifferentiated,unicellular state. The new plant cell's genetic composition andphenotype is a technical effect created by the integration of theheterologous DNA into the genome of the cell. Another aspect of thepresent invention is a method of using a microorganism of the presentinvention. Methods of using microorganisms of the present invention,such as transgenic plant cells, include (i) methods of producingtransgenic cells by integrating recombinant DNA into genome of the celland then using this cell to derive additional cells possessing the sameheterologous DNA; (ii) methods of culturing cells that containrecombinant DNA using modern microbiology techniques; (iii) methods ofproducing and purifying endogenous or recombinant carbohydrate, lipid,nucleic acid, or protein products from cultured cells; and (iv) methodsof using modern plant tissue culture techniques with transgenic plantcells to produce transgenic plants or transgenic plant tissue cultures.

As used herein, “progeny” includes any plant, seed, plant cell, and/orregenerable plant part comprising the event DNA derived from an ancestorplant and/or a polynucleotide having at least one of the sequencesprovided as SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 7, or SEQ ID NO: 8.Plants, progeny, and seeds may be homozygous or heterozygous for thetransgene. Progeny may be grown from seeds produced by a plantcomprising event MON 88302 and/or from seeds produced by a plantfertilized with pollen from a plant comprising event MON 88302.

Progeny plants may be self-pollinated (also known as “selfing”) togenerate a true breeding line of plants, i.e., plants homozygous for thetransgene. Selfing of appropriate progeny can produce plants that arehomozygous for both added, exogenous genes.

Alternatively, progeny plants may be outcrossed, e.g., bred with anotherplant, to produce a varietal or a hybrid seed or plant. The other plantmay be transgenic or nontransgenic. A varietal or hybrid seed or plantof the present invention may thus be derived by crossing a first parentthat lacks the specific and unique DNA of the event MON 88302 with asecond parent comprising event MON 88302, resulting in a hybridcomprising the specific and unique DNA of the event MON 88302. Eachparent can be a hybrid or an inbred/varietal, so long as the cross orbreeding results in a plant or seed of the present invention, i.e., aseed having at least one allele containing the specific and unique DNAof event MON 88302 and/or SEQ ID NO: 1 and SEQ ID NO: 2. Two differenttransgenic plants may thus be mated to produce hybrid offspring thatcontain two independently segregating, added, exogenous genes. Forexample, the glyphosate tolerant plant comprising event MON 88302 can becrossed with another transgenic plant to produce a plant having thecharacteristics of both transgenic parents. One example of this would bea cross of glyphosate tolerant Brassica plant comprising event MON 88302with a Brassica plant such as mustard or canola and having one or moreadditional traits, resulting in a progeny plant or seed that is tolerantto glyphosate and has one or more additional traits. Brassica plantshaving desirable transgenic traits are known in the art, including butnot limited to Brassica plants having the trait of herbicide tolerance(e.g., event RT200, event RT73, event MS1, event RF1, event RF2, Topas19/2, MS8, RF3, T45), a hybrid breeding system or a fertility system(e.g., event MS1, event MS8, event RF1, event RF2, event Rf3), insectcontrol, enhanced yield, disease resistance (e.g., SclerotiniaResistance, Blackleg Resistance, Clubroot Resistance, Fusarium WiltResistance), altered or enhanced oil composition (e.g., eventpCGN3828-212/86-18, event pCGN3828-212/23), all described for example inthe publicly available United States Department of Agriculture (USDA)Animal and Plant Health Inspection Service (APHIS) listing of Petitionsfor Nonregulated Status. Brassica plants having desirable non-transgenictraits are known in the art, including but not limited to traits forherbicide tolerance (e.g., 1471 for imidazolinone tolerance, CLB-1 forimidazolinone tolerance, TTC for triazine tolerance), pathogenresistance, insect control, enhanced yield, disease resistance (e.g.,Sclerotinia Resistance, Blackleg Resistance, Clubroot Resistance,Fusarium Wilt Resistance), altered or enhanced oil composition(including low linolenic and/or high oleic), altered chemical and/ornutritional composition, altered protein composition, cold tolerance,drought tolerance, altered maturity and/or flowering, and other alteredor improved agronomic qualities.

Back-crossing to a parental plant and out-crossing with a non-transgenicplant are also contemplated, as is vegetative propagation. Descriptionsof other breeding methods that are commonly used for different traitsand crops can be found in one of several references, e.g., Fehr, inBreeding Methods for Cultivar Development, Wilcox J. ed., AmericanSociety of Agronomy, Madison Wis. (1987).

The present invention provides a plant part that is derived from plantscomprising event MON 88302. As used herein, a “plant part” refers to anypart of a plant which is comprised of material derived from a plantcomprising event MON 88302. Plant parts include but are not limited topollen, ovule, pod, flower, root or stem tissue, fibers, and leaves.Plant parts may be viable, nonviable, regenerable, and/ornon-regenerable.

The present invention provides a commodity product that is derived froma plant comprising event MON 88302. As used herein, a “commodityproduct” refers to any composition or product which is comprised ofmaterial derived from a plant, seed, plant cell, or plant partcomprising event MON 88302. Commodity products may be sold to consumersand may be viable or nonviable. Nonviable commodity products include butare not limited to nonviable seeds and grains; processed seeds, seedparts, and plant parts; dehydrated plant tissue, frozen plant tissue,and processed plant tissue; seeds and plant parts processed for animalfeed for terrestrial and/or aquatic animals consumption, oil, meal,flour, flakes, bran, fiber, and any other food for human consumption;and biomasses and fuel products. Viable commodity products include butare not limited to seeds and plant cells. A plant comprising event MON88302 can thus be used to manufacture any commodity product typicallyacquired from a Brassica plant. Any such commodity product that isderived from the plants comprising event MON 88302 may contain at leasta detectable amount of the specific and unique DNA corresponding toevent MON 88302, and specifically may contain a detectable amount of apolynucleotide containing at least 40 contiguous nucleotides of SEQ IDNO: 1 or SEQ ID NO: 2. Any standard method of detection forpolynucleotide molecules may be used, including methods of detectiondisclosed herein. A commodity product is within the scope of the presentinvention if there is any detectable amount of SEQ ID NO: 1 or SEQ IDNO: 2 in the commodity product.

The plants, progeny, seeds, plant cells, plant parts (such as pollen,ovule, pod, flower, root or stem tissue, and leaves), and commodityproducts of the present invention are therefore useful for, among otherthings, growing plants for the purpose of producing seed and/or plantparts comprising event MON 88302 for agricultural purposes, producingprogeny comprising event MON 88302 for plant breeding and researchpurposes, use with microbiological techniques for industrial andresearch applications, and sale to consumers.

The present invention provides methods for controlling weeds in a field.A method for controlling weeds in a field is provided that consists ofplanting plants comprising event MON 88302 in a field and applying atleast one herbicidally effective dose of glyphosate to the field for thepurpose of controlling weeds in the field without injuring plantscomprising MON 88302. Another method for controlling weeds in a field isalso provided that consists of applying at least one herbicidallyeffective dose of glyphosate to the field to control weeds in the fieldand then planting crops comprising event MON 88302 in the field. Themethods of the invention may be used alone or in combination.Application of glyphosate may be pre-plant (i.e., anytime prior toplanting seed comprising event MON 88302 including, but not limited to,about 14 days pre-planting to about 1 day pre-planting or concurrentwith sowing seed comprising event MON 88302), pre-emergence (i.e., anytime after seed comprising event MON 88302 is planted and before plantscomprising event MON 88302 emerge), and/or post-emergence (i.e., anytime after plants comprising event MON 88302 emerge). In practicing themethods of the invention, multiple applications of glyphosate may beused over a growing season, for example, as two applications (such as apre-planting application and a post-emergence application or apre-emergence application and a post-emergence application) or threeapplications (such as a pre-planting application, a pre-emergenceapplication, and a post-emergence application). The total glyphosateapplied over the growing season may thus include one or more in-seasonapplication where the sum of multiple in-season applications addstogether to make the total glyphosate applied. As used herein, an amountof glyphosate effective to control the growth of weeds, i.e., anherbicidally effective dose of glyphosate for use in the field as anin-crop application to control the growth of weeds in the filed, shouldconsist of a range from about 0.125 pounds of glyphosate per acre toabout 6.4 pounds of glyphosate per acre total over a growing season. Forexample, an herbicidally effective dose of glyphosate for use in thefield as an in-crop application may be at least about 0.125, about 0.5,about 1.0, about 1.6, about 2.0, about 2.5, about 3.0, about 3.5, about4.0, about 4.5, about 5.0, about 5.5, about 6.0, or about 6.4 pounds peracre total over a growing season.

Methods for producing an herbicide tolerant plant comprising transgenicevent MON 88302 are provided. Transgenic plants used in these methodsmay be homozygous or heterozygous for the transgene. Progeny plantsproduced by these methods may be varietal or hybrid plants; may be grownfrom seeds produced by a plant and/or from seed comprising event MON88302 produced by a plant fertilized with pollen from a plant comprisingevent MON 88302; and may be homozygous or heterozygous for thetransgene. Progeny plants may be subsequently self-pollinated togenerate a true breeding line of plants, i.e., plants homozygous for thetransgene, or alternatively may be outcrossed, e.g., bred with anotherunrelated plant, to produce a varietal or a hybrid seed or plant.

A plant that tolerates application of glyphosate herbicide may beproduced by sexually crossing a plant comprising event MON 88302comprising a polynucleotide molecule comprising the sequence of SEQ IDNO: 1 and SEQ ID NO: 2 with another plant and thereby producing seed,which is then grown into progeny plants. These progeny plants may thenbe treated with glyphosate herbicide to select for progeny plants thatare tolerant to glyphosate herbicide. Alternatively, these progenyplants may be analyzed using diagnostic methods to select for progenyplants that contain the event MON 88302 DNA. The other plant used in thecrossing may or may not be tolerant to glyphosate herbicide and may ormay not be transgenic. The progeny plant and/or seed produced may bevarietal or hybrid seed. In practicing this method, the step of sexuallycrossing one plant with another plant, i.e., cross-pollinating, may beaccomplished or facilitated by human intervention, for example: by humanhands collecting the pollen of one plant and contacting this pollen withthe style or stigma of a second plant; by human hands and/or humanactions removing, destroying, or covering the stamen or anthers of aplant (e.g., by manual intervention or by application of a chemicalgametocide) so that natural self-pollination is prevented andcross-pollination would have to take place in order for fertilization tooccur; by human placement of pollinating insects in a position for“directed pollination” (e.g., by placing beehives in orchards or fieldsor by caging plants with pollinating insects); by human opening orremoving of parts of the flower to allow for placement or contact offoreign pollen on the style or stigma; by selective placement of plants(e.g., intentionally planting plants in pollinating proximity); and/orby application of chemicals to precipitate flowering or to fosterreceptivity (of the stigma for pollen).

A plant that tolerates application of glyphosate herbicide may beproduced by selfing a plant comprising event MON 88302 comprising apolynucleotide molecule comprising the sequence of SEQ ID NO: 1 and SEQID NO: 2 and thereby producing seed, which is then grown into progenyplants. These progeny plants may then be treated with glyphosateherbicide to select for progeny plants that are tolerant to glyphosateherbicide. Alternatively, these progeny plants may be analyzed usingdiagnostic methods to select for progeny plants that contain the eventMON 88302 DNA. In practicing this method, the step of sexually crossingone plant with itself, i.e., self-pollinating or selfing, may beaccomplished or facilitated by human intervention, for example: by humanhands collecting the pollen of the plant and contacting this pollen withthe style or stigma of the same plant and then optionally preventingfurther fertilization of the plant; by human hands and/or actionsremoving, destroying, or covering the stamen or anthers of other nearbyplants (e.g., by detasseling or by application of a chemical gametocide)so that natural cross-pollination is prevented and self-pollinationwould have to take place in order for fertilization to occur; by humanplacement of pollinating insects in a position for “directedpollination” (e.g., by caging a plant alone with pollinating insects);by human manipulation of the flower or its parts to allow forself-pollination; by selective placement of plants (e.g., intentionallyplanting plants beyond pollinating proximity); and/or by application ofchemicals to precipitate flowering or to foster receptivity (of thestigma for pollen).

Progeny plants and seeds encompassed by these methods and produced byusing these methods will be distinct from other plants, for examplebecause the progeny plants and seeds: are recombinant and as suchcreated by human intervention; are glyphosate herbicide tolerant;contain at least one allele that consists of the transgene DNA of thepresent invention; and/or contain a detectable amount of apolynucleotide sequence selected from the group consisting of SEQ ID NO:1 and SEQ ID NO: 2. A seed may be selected from an individual progenyplant, and so long as the seed comprises SEQ ID NO: 1 and SEQ ID NO: 2,it will be within the scope of the present invention.

In practicing the present invention, two different transgenic plants canbe crossed to produce hybrid offspring that contain two independentlysegregating heterologous genes. Selfing of appropriate progeny canproduce plants that are homozygous for both genes. Back-crossing to aparental plant and out-crossing with a non-transgenic plant are alsocontemplated, as is vegetative propagation. Descriptions of othermethods that are commonly used for different traits and crops can befound in one of several references, e.g., Fehr, in Breeding Methods forCultivar Development, Wilcox J. ed., American Society of Agronomy,Madison Wis. (1987).

The plants and seeds used in the methods disclosed herein may alsocontain one or more additional transgenes. Such transgene may be anynucleotide sequence encoding a protein or RNA molecule conferring adesirable trait including but not limited to increased insectresistance, increased water use efficiency, increased yield performance,increased drought resistance, increased seed quality, improvednutritional quality, and/or increased herbicide tolerance, in which thedesirable trait is measured with respect to a plant lacking suchadditional transgene.

The methods of the present invention are therefore useful for, amongother things, controlling weeds in a field while growing plants for thepurpose of producing seed and/or plant parts comprising event MON 88302for agricultural or research purposes, selecting for progeny comprisingevent MON 88302 for plant breeding or research purposes, and producingprogeny plants and seeds comprising event MON 88302.

The plants, progeny, seeds, plant cells, plant parts (such as pollen,ovule, pod, flower, root or stem tissue, and leaves), and commodityproducts of the present invention may be evaluated for DNA composition,gene expression, and/or protein expression. Such evaluation may be doneby using standard methods such as PCR, northern blotting, southernanalysis, western blotting, immuno-precipitation, and ELISA or by usingthe methods of detection and/or the detection kits provided herein.

Methods of detecting the presence of materials specific to event MON88302 in a sample are provided. One method consists of detecting thepresence of DNA specific to and derived from a cell, tissue, or plantcomprising event MON 88302. The method provides for a template DNAsample to be contacted with a primer pair that is capable of producingan amplicon from event MON 88302 DNA upon being subjected to conditionsappropriate for thermal amplification, particularly an amplicon thatcontains at least 40 contiguous nucleotides of either SEQ ID NO: 1 orSEQ ID NO: 2 or the complements thereof. The amplicon is produced from atemplate DNA molecule derived from event MON 88302, so long as thetemplate DNA molecule incorporates the specific and unique nucleotidesequences as set forth in SEQ ID NO: 1 and SEQ ID NO: 2. The ampliconmay be single or double stranded DNA or RNA, depending on the polymeraseselected for use in the production of the amplicon. The method providesfor detecting the amplicon molecule produced in any such thermalamplification reaction, and confirming within the sequence of theamplicon the presence of the nucleotides corresponding to SEQ ID NO: 1or SEQ ID NO: 2 or the complements thereof. The detection of thenucleotides corresponding to SEQ ID NO: 1 or SEQ ID NO: 2 or thecomplements thereof in the amplicon are determinative and/or diagnosticfor the presence of event MON 88302 specific DNA and thus biologicalmaterial comprising event MON 88302 in the sample.

Another method is provided for detecting the presence of a DNA moleculecorresponding to SEQ ID NO: 3 and SEQ ID NO: 4 in a sample consisting ofmaterial derived from plant or plant tissue. The method consists of (i)extracting a DNA sample from a plant, or from a group of differentplants, (ii) contacting the DNA sample with a DNA probe molecule thatexhibits at least 40 contiguous nucleotides as set forth in either SEQID NO: 1 or SEQ ID NO: 2, (iii) allowing the probe and the DNA sample tohybridize under stringent hybridization conditions, and then (iv)detecting a hybridization event between the probe and the target DNAsample. Detection of the hybrid composition is diagnostic for thepresence of SEQ ID NO: 3 or SEQ ID NO: 4, as the case may be, in the DNAsample. Absence of hybridization is alternatively diagnostic of theabsence of the transgenic event in the sample. Alternatively,determining that a particular plant contains either or both of thesequences corresponding to SEQ ID NO: 1 or SEQ ID NO: 2, or thecomplements thereof, is determinative that the plant contains at leastone allele corresponding to the event MON 88302.

It is thus possible to detect the presence of a nucleic acid molecule ofthe present invention by any well known nucleic acid detection methodsuch as the polymerase chain reaction (PCR) or DNA hybridization usingnucleic acid probes. An event-specific PCR assay is discussed, forexample, by Taverniers et al. (J. Agric. Food Chem., 53: 3041-3052,2005) in which an event-specific tracing system for transgenic maizelines BO 1, Bt176, and GA21 and for transgenic event RT73 isdemonstrated. In this study, event-specific primers and probes weredesigned based upon the sequences of the genome/transgene junctions foreach event. Transgenic plant event specific DNA detection methods havealso been described in U.S. Pat. Nos. 6,893,826; 6,825,400; 6,740,488;6,733,974; 6,689,880; 6,900,014 and 6,818,807.

DNA detection kits are provided. One type of kit contains at least oneDNA molecule of sufficient length of contiguous nucleotides of SEQ IDNO: 3, SEQ ID NO: 5, or SEQ ID NO: 6 to function as a DNA primer orprobe specific for detecting the presence of DNA derived from transgenicevent MON 88302 in a sample. The DNA molecule being detected with thekit contains at least 40 contiguous nucleotides of the sequence as setforth in SEQ ID NO: 1. Alternatively, the kit may contain at least oneDNA molecule of sufficient length of contiguous nucleotides of SEQ IDNO: 4, SEQ ID NO: 5, or SEQ ID NO: 6 to function as a DNA primer orprobe specific for detecting the presence of DNA derived from transgenicevent MON 88302 in a sample. The DNA molecule being detected with thekit contains at least 40 contiguous nucleotides as set forth in SEQ IDNO: 2.

An alternative kit employs a method in which the target DNA sample iscontacted with a primer pair as described above, then performing anucleic acid amplification reaction sufficient to produce an ampliconcomprising at least 40 contiguous nucleotides of SEQ ID NO: 1 or SEQ IDNO: 2. Detection of the amplicon and determining the presence of nofewer than 40 contiguous nucleotides of SEQ ID NO: 1 or SEQ ID NO: 2 orthe complements thereof within the sequence of the amplicon isdiagnostic for the presence of event MON 88302 specific DNA in a DNAsample.

A DNA molecule sufficient for use as a DNA probe is provided that isuseful for determining, detecting, or for diagnosing the presence oreven the absence of DNA specific and unique to event MON 88302 DNA in asample. The DNA molecule contains at least 40 contiguous nucleotides ofSEQ ID NO: 1, or the complement thereof, or at least 40 contiguousnucleotides of SEQ ID NO: 2, or the complement thereof.

Nucleic-acid amplification can be accomplished by any of the variousnucleic-acid amplification methods known in the art, including thermalamplification methods. The sequence of the heterologous DNA insert,junction sequences, or flanking sequences from event MON 88302 (withrepresentative seed samples comprising event MON 88302 deposited as ATCCPTA-10955) can be verified (and corrected if necessary) by amplifyingsuch sequences from the event using primers derived from the sequencesprovided herein followed by standard DNA sequencing of the amplicon orof the cloned DNA.

The amplicon produced by these methods may be detected by a plurality oftechniques. One such method is Genetic Bit Analysis (Nikiforov, et al.Nucleic Acid Res. 22:4167-4175, 1994) where a DNA oligonucleotide isdesigned which overlaps both the adjacent flanking genomic DNA sequenceand the inserted DNA sequence. The oligonucleotide is immobilized inwells of a microwell plate. Following thermal amplification of theregion of interest (using one primer in the inserted sequence and one inthe adjacent flanking genomic sequence), a single-stranded amplicon canbe hybridized to the immobilized oligonucleotide and serve as a templatefor a single base extension reaction using a DNA polymerase and labelledddNTPs specific for the expected next base. Readout may be fluorescentor ELISA-based. Detection of a fluorescent or other signal indicates thepresence of the insert/flanking sequence due to successfulamplification, hybridization, and single base extension.

Another method is the Pyrosequencing technique as described by Winge(Innov. Pharma. Tech. 00:18-24, 2000). In this method an oligonucleotideis designed that overlaps the adjacent genomic DNA and insert DNAjunction. The oligonucleotide is hybridized to a single-strandedamplicon from the region of interest (one primer in the insertedsequence and one in the flanking genomic sequence) and incubated in thepresence of a DNA polymerase, ATP, sulfurylase, luciferase, apyrase,adenosine 5′ phosphosulfate and luciferin. ddNTPs are added individuallyand the incorporation results in a light signal which is measured. Alight signal indicates the presence of the transgene insert/flankingsequence due to successful amplification, hybridization, and single ormulti-base extension.

Fluorescence Polarization as described by Chen, et al., (Genome Res.9:492-498, 1999) is a method that can be used to detect the amplicon.Using this method an oligonucleotide is designed which overlaps thegenomic flanking and inserted DNA junction. The oligonucleotide ishybridized to single-stranded amplicon from the region of interest (oneprimer in the inserted DNA and one in the flanking genomic DNA sequence)and incubated in the presence of a DNA polymerase and afluorescent-labeled ddNTP. Single base extension results inincorporation of the ddNTP. Incorporation can be measured as a change inpolarization using a fluorometer. A change in polarization indicates thepresence of the transgene insert/flanking sequence due to successfulamplification, hybridization, and single base extension.

TAQMAN® (PE Applied Biosystems, Foster City, Calif.) may also be used todetect and/or quantifying the presence of a DNA sequence using theinstructions provided by the manufacturer. Briefly, a FREToligonucleotide probe is designed which overlaps the genomic flankingand insert DNA junction. The FRET probe and amplification primers (oneprimer in the insert DNA sequence and one in the flanking genomicsequence) are cycled in the presence of a thermostable polymerase anddNTPs. Hybridization of the FRET probe results in cleavage and releaseof the fluorescent moiety away from the quenching moiety on the FRETprobe. A fluorescent signal indicates the presence of theflanking/transgene insert sequence due to successful amplification andhybridization.

Molecular Beacons have been described for use in sequence detection asdescribed in Tyangi, et al. (Nature Biotech. 14:303-308, 1996). Briefly,a FRET oligonucleotide probe is designed that overlaps the flankinggenomic and insert DNA junction. The unique structure of the FRET proberesults in it containing secondary structure that keeps the fluorescentand quenching moieties in close proximity. The FRET probe andamplification primers (one primer in the insert DNA sequence and one inthe flanking genomic sequence) are cycled in the presence of athermostable polymerase and dNTPs. Following successful amplification,hybridization of the FRET probe to the target sequence results in theremoval of the probe secondary structure and spatial separation of thefluorescent and quenching moieties resulting in the production of afluorescent signal. The fluorescent signal indicates the presence of theflanking/transgene insert sequence due to successful amplification andhybridization.

Other described methods, such as, microfluidics (US Patent PublicationNo. 2006068398, U.S. Pat. No. 6,544,734) provide methods and devices toseparate and amplify DNA samples. Optical dyes used to detect andmeasure specific DNA molecules (WO/05017181). Nanotube devices(WO/06024023) that comprise an electronic sensor for the detection ofDNA molecules or nanobeads that bind specific DNA molecules and can thenbe detected.

DNA detection kits can be developed using the compositions disclosedherein and the methods well known in the art of DNA detection. The kitsare useful for the identification of event MON 88302 in a sample and canbe applied to methods for breeding plants containing the appropriateevent DNA. The kits may contain DNA primers or probes that are similaror complementary to SEQ ID NO: 1-6, or fragments or complements thereof.

The kits and detection methods of the present invention are thereforeuseful for, among other things, identifying event MON 88302, selectingplant varieties or hybrids comprising event MON 88302, detecting thepresence of DNA derived from the event MON 88302 in a sample, andmonitoring samples for the presence and/or absence of event MON 88302 orplants, plant parts or commodity products comprising event MON 88302.

The following examples are included to demonstrate examples of certainpreferred embodiments of the invention. It should be appreciated bythose of skill in the art that the techniques disclosed in the examplesthat follow represent approaches the inventors have found function wellin the practice of the invention, and thus can be considered toconstitute examples of preferred modes for its practice. However, thoseof skill in the art should, in light of the present disclosure,appreciate that many changes can be made in the specific embodimentsthat are disclosed and still obtain a like or similar result withoutdeparting from the spirit and scope of the invention.

EXAMPLES Example 1 Transformation of Brassica napus and MON 88302 EventSelection

This example describes how transgenic events were created and how eventMON 88302 was selected. The transgenic event MON 88302 was generated byAgrobacterium-mediated transformation of Brassica napus cells with thetransgenic DNA illustrated in FIG. 1, the sequence of which is set forthin SEQ ID NO: 5. The transgene insert provided as SEQ ID NO: 5 is anexpression cassette comprising a chimeric promoter consisting of: anenhancer molecule derived from 35S enhancer from figwort mosaic virus(E-FMV.35S) operably linked to a promoter molecule derived from derivedfrom Arabidopsis thaliana Tsf1 gene (P-At.Tsf1) operably linked to aleader molecule derived from Arabidopsis thaliana Tsf1 gene (L-At.Tsf1);operably linked to an intron sequence derived from Arabidopsis thalianaTsf1 gene (I-At.Tsf1); operably linked to a DNA molecule encoding achloroplast transit peptide (CTP2, Arabidopsis thaliana EPSPS); operablylinked to a DNA molecule encoding a glyphosate resistant EPSPS(CP4-syn); operably connected to a 3′ UTR DNA molecule derived fromPisum sativum gene (T-RbcS2, E9).

Explants from Brassica napus were first each transformed with one offive expression cassettes using Agrobacterium-mediated transformation.Transformed cells were then selected on media containing glyphosate andsurviving cells were regenerated into plants. Over 23,000 explants weregenerated and from this 224 total individual R0 events were produced andused for subsequent screening (Table 1).

TABLE 1 Transformation of Brassica napus and MON 88302 event selectionSin- GH GH FT FT con- transfor- gle effi- molec- First Second Leadstruct mation copy cacy ular year year Events 1 97 events 37 24  16  16 13 3 from 8325 advanced explants 8 tested 2 69 events 32 5 3 3 0 — from4625 explants 3 43 events 11 0 — — — — from 10245 explants 4 ~30 events14 6 NA 3 0 — 5 ~25 events 10 6 NA 3 0 —

Tissue samples of the events were screened using TAQMAN® PCR analysis toeliminate multi-copy and/or molecularly complex events. From the initial224 events, 104 were advanced based on the presence of a single copy ofthe transgene and the absence of vector backbone sequences as determinedby TAQMAN® PCR analysis. Specifically, from construct 1 (with theinitial 97 events) 37 R1 events were advanced; from construct 2 (withthe initial 69 events) 32 R1 events were advanced; from construct 3(with the initial 43 events) 11 R1 events were advanced; from construct4 (with approximately 30 events) 14 R1 events were advanced, and fromconstruct 5 (with approximately 25 events) 10 R1 events were advanced.

The 104 events containing a single copy of the transgene were advancedto greenhouse testing (GH), including additional molecular analysis andefficacy testing. R1 greenhouse molecular analysis included: an initialSouthern blot analysis to confirm the presence of a single copy of thetransgene and the absence of vector backbone sequences; CP4 proteinexpression levels measured at the 3-4 leaf stage, the rosette stage andin the seed; and CP4 processing as measured by Western blot analysis(events from constructs 4 and 5 were not included in this analysis). R1greenhouse efficacy testing included: segregation, Chlorosis/Necrosis,vegetative tolerance, first flower, growth reduction, male reproductivetolerance as measured by pollen viability, and seed counts. Fromanalysis of the greenhouse molecular and efficacy testing for the 104events, 25 events were advanced to first year field trials.Specifically, for construct 1 there were 16 R2 events from the 37 R1events tested that were advanced; for construct 2 there were 3 R2 eventsfrom the 32 R1 events tested that were advanced; for construct 3, with11 R1 events advanced to greenhouse testing, 0 R2 events were advanced;for construct 4 there were 3 R2 events from the 14 R1 events tested thatwere advanced; for construct 5 there were 3 R2 events from the 10 R1events tested that were advanced to field testing.

The 25 R2 events were then analyzed in first year field testing foragronomic evaluation in paired plots at the same locations. Agronomicfield trials were initiated to evaluate the impact of gene insertion onplant growth and development. The 16 R2 events from construct 1 wereevaluated at 5 locations. The 3 R2 events from construct 2, the 3 R2events from construct 4, and the 3 R2 events from construct 5 wereevaluated at 4 locations. Each study contained the positive and negativeisoline pair of each event, as well as the parental transformationgermplasm and a commercial variety. For agronomic evaluation, plantvigor, early stand, date of first flower, and plant height were observedto assess plant development. Yield data were also evaluated as anindication of gene insertional effect on plant growth and development. Apaired split plot design with 3 replications was used with event aswhole plots and isolines as subplots. Positive and negative isolines ofeach event were paired while a commercial variety and the transformationbackground were paired and included as controls. Plots were maintainedweed-free throughout the season employing conventional herbicideprograms supplemented by hand-weeding; as necessary. No glyphosatetreatments were applied in this field protocol.

An emergence/stand count for each plot was taken at 7 to 10 days afterplanting. Cotyledons that had completely cleared the soil wereconsidered “emerged”. Plant vigor was determined at the 2 to 3 leafstage (prior to the first herbicide application of the paired plot ofthe efficacy testing) using a scale ranging between 1 (excellent vigor),with 5 being average, and 9 (poor vigor). The date of first flower wasrecorded after all plots had begun flowering and was expressed aspercentage of open flowers per plant per plot. Plant height of 5 plantsper plot was measured from the soil surface to the top of highest racemeat mid to late flower and expressed in cm. Uniformity and standabilitywere estimated using a scale ranging between 1 (excellent), with 5 beingaverage, and 9 (poor). Each trial was harvested after majority of plotshad reached maturity. Some plots were directly combined while otherplots were pushed once maturity was reached and the swath was combinedapproximately 10 days thereafter. Moisture and individual seed weightswere recorded (lb/A) and a seed samples were collected for oil andprotein analysis. Each location was analyzed individually and averagesacross locations were analyzed using statistical software. Data werescreened for outliers using the standard two-pass procedure based ondeleted studentized residuals using a Bonferroni adjustment for anexperiment-wise Type I error rate of 5% at each location. Outliers wereremoved prior to analysis. The standard analysis of variance for asplit-plot design was performed with restricted maximum likelihoodestimation. Least-squares means were calculated for each isoline of eachevent, and t-tests with comparison-wise error rates of 5% were used todetermine the significance of gene insertion on canola growthcharacteristics. Event traits of positive isolines were compared to thetraits of the corresponding negative isolines. Due to differences inmaturity between events, comparison between positive isolines and thepooled negative isolines as well as comparisons to the commercialstandard and transformation background were excluded.

Results from first year agronomy field trials indicated that both of thetwo constructs produced multiple events in which agronomiccharacteristics and yield were not negatively impacted by geneinsertion. Based on agronomic evaluation, 15 events were candidates tobe advanced to second year field trials. Specifically, 14 events fromconstruct 1 (16 R2 events in field trials) and 1 event from construct 2(3 R2 events in field trials) were candidates to be advanced to secondyear field trials.

The 25 events were also evaluated in first year field testing forefficacy located at the same testing site as the agronomy field trials.Efficacy field trials were initiated to evaluate the vegetative andreproductive tolerance of the events to sequential applications ofglyphosate at various application timings and rates. The 16 R2 eventsfrom construct 1 were evaluated at 5 locations. The 3 R2 events fromconstruct 2, the 3 R2 events from construct 4, and the 3 R2 events fromconstruct 5 were evaluated at 4 locations. Each study contained thepositive isoline of each event and two commercial varieties. A splitplot design with 3 replications was used with herbicide treatments aswhole plots and event entries as subplots. A non-sprayed treatment wasincluded for comparison.

Events were evaluated for vegetative and reproductive tolerance toeither a single application of 1.6 lb AE/A or two sequentialapplications of 0.8 lb AE/A when applied from crop emergence up to firstflower. Roundup WeatherMAX® was applied at three different rates thatwere equivalent to 2×, 4×, and 8× of the single application rate (Table2). The initial application was made at the four leaf stage followed bya second application at the prebolt stage. To minimize potential plantdamage from surfactants and other formulation components, spraysolutions to deliver 3.2 and 6.4 lb AE/A (4× and 8×) were prepared using1.6 lb AE/A of Roundup WeatherMAX® as base rate and adding technicalmaterial (glyphosate without surfactant) to achieve the desired rate.All applications were made using a tractor mounted spray boom equippedwith flat-fan nozzles calibrated to deliver 10 to 20 GPA.

Data Collection was performed as above. Glyphosate injury was assessedas follows. Prior to each glyphosate application, plant height andnumber of leaves for 5 plants per plot in the treated and non-treatedplots were documented. To determine if glyphosate caused injury to theevents, visual estimates of percent chlorosis and percent necrosis weretaken at 7 to 10 days after each glyphosate application. Vegetativeinjury was rated after the first glyphosate application whilereproductive percent chlorosis/necrosis was evaluated after the secondapplication using a scale ranging from 0% (no injury) to 100% (plantdeath). Growth reduction was evaluated at 14 to 21 days after eachapplication using a scale ranging from 0 to 100%. Vegetative growthreduction was evaluated after the first application while reproductivegrowth reduction was determined after the sequential application.Least-squares means were calculated for each combination of glyphosatelevel and event, and t-tests were used to determine the significance ofthe effects of glyphosate on event tolerance. Event yields fromglyphosate treatments were compared to the yield of the non-treatedcontrol as well as to the commercial standard, RT73, within eachherbicide rate, and comparisons across herbicide rates were made foreach event.

Results from the first year efficacy field trials indicate thatconstruct 1 produced events which had excellent vegetative andreproductive tolerance to glyphosate. Vegetative injury observed at the2× product concept rate was very minor and considered to be commerciallyinsignificant. Outstanding reproductive tolerance for construct 1 eventswas observed. Yield of all construct 1 events was not negativelyimpacted by glyphosate, regardless of rate, with the exception of oneevent which showed a significant yield reduction at the highestapplication rate (6.4 lb AE/A followed by 6.4 lb AE/A). Events derivedfrom construct 2 did not have sufficient vegetative glyphosate tolerancefor advancement. Unacceptable levels of injury were observed at allglyphosate rates tested. Glyphosate significantly reduced the yield ofall construct 2 events, regardless of application rate. Events derivedfrom construct 4 and 5 did not perform as well as events from construct1 and therefore were not advanced to second year field testing. Based onthe first year field testing, 13 events from construct 1 were advancedto second year field testing.

Example 2 Comparison of RT73 to MON 88302

Field trials were then designed to evaluate MON 88302 compared to thecurrent commercial Genuity™ Roundup Ready® Canola (RT73 event).Comparisons of MON 88302 to RT73 showed that MON 88302 provided superiorcrop tolerance to higher glyphosate application rates thus enablingimproved weed control at higher glyphosate rates of hard to controlweeds such as dandelion, Canada thistle, foxtail barley, wild buckwheat,common-lambsquarter, and kochia. Comparisons of MON 88302 to RT73 alsoshowed that MON 88302 provided superior crop tolerance to a widerapplication range for glyphosate thus enabling glyphosate applicationover a wider window ranging from post emergence to first flower,enabling yield-robbing weed control even at a later growth stage of thecrop than was possible with RT73. The MON 88302 combination of anincreased tolerance to higher glyphosate rates and tolerance toglyphosate applications at a later crop stage provides the advantage, ascompared to RT73, of allowing for application of glyphosate at a latergrowth stage when environmental conditions have limited earlyapplications and/or improved control of late flushes of weeds that wouldreduce crop yield.

The current registered glyphosate rate for the Genuity™ Roundup Ready®Canola system (event RT73) is a single application of 675 g ae/ha or two450 g ae/ha applications up to the six leaf stage of the canola crop.RT73 was compared with the MON 88302 event at multiple singleapplication rates of glyphosate. Applications were made from postemergence to the first flower of the canola crop. The Genuity™ RoundupReady® Canola system (RT73 event) was represented by the commercial openpollinated variety 34-65, and event MON88302 was transformed into theEbony germplasm.

Eight trials were established with six taken to harvest (one site waslost to drought and one site was lost to hail, and data from oneharvested site was not used due to the coefficient of variation (CV)exceeding the predetermined cut-off value). Standard canola growingpractices were utilized throughout the season to optimize plant growth.Pre-emergent and post-emergent conventional herbicides and seedtreatment containing fungicide and insecticide were used to minimizepest pressure. Trials were set up as a split block design with Genuity™Roundup Ready® Canola (RT73 event) or MON 88302 systems blocked andherbicide rates randomized within the block. Plots were two by sixmeters and replicated three times. Plots were sprayed with handheld boomsprayers at 100-110 l/ha application rate at the appropriate crop stage.The Canadian formulation of Roundup WeatherMAX® (540 g/L) was used asthe glyphosate product. The four to six leaf stage was defined as fourto six true leaves on the main stem, and first flower was when 50% ofthe plants had at least one flower. Percent chlorosis (% CHLR) wasrecorded seven to ten days after herbicide application (DAT or daysafter treatment). Percent chlorosis is a visual estimate of the amountof yellowing on the leaves of plants as a result of herbicide treatmentcompared to the untreated check on a 1 to 100 scale. Percent growthreduction (% GR) was recorded 14-21 days after herbicide application(DAT). Percent growth reduction is a visual evaluation used to describeplant growth reduction consisting of, but not limited to, reduced heightand/or quantity of foliage for the treated area. The evaluation pertainsonly to the above ground portion of the plant. Percent growth reductionis compared to the non-treated check and is rated on a 1 to 100 scale.Maturity was recorded as days after planting to when 30% of the seeds onthe main raceme are brown/black in color. Percent seed moisture wasrecorded electronically during harvesting. All plots were swathed andthen allowed to dry until seed moisture was low enough to facilitateharvesting. Weight and seed moisture was recorded electronically duringharvesting and converted to bushels/acre at 10% seed moisture. Eachlocation was analyzed individually and averages across locations wereanalyzed using statistical software. Data were screened for outliersusing the standard two-pass procedure based on deleted studentizedresiduals using a False Discovery Rate (FDR) adjustment for anexperiment-wise Type I error rate of 5% at each location. Outliers wereremoved prior to analysis. The standard analysis of variance for asplit-plot design was performed by mixed model with restricted maximumlikelihood estimation-variety and herbicide treatments were treated asfixed effects, replications and locations were random effects.Least-squares means were calculated for each variety and each treatment,and t-tests with comparison-wise error rates of 5% were used todetermine the significance between variety and control at each herbicidetreatment level on canola growth characteristics. Crop injury, maturity,and yield were measured to assess benefits.

Data on the glyphosate tolerance of canola comprising event RT73compared to canola comprising event MON 88302 are provided in Table 2.Briefly, the glyphosate tolerance of MON 88302 was superior to theglyphosate tolerance of RT73 in both the tolerance of increasedglyphosate application rates (g ae/ha) and the range of crop stage whereglyphosate application was tolerated. For example, RT73 showed 4.7%chlorosis at the 1800 g ae/ha application rate to the 4-6 leaf stage,where MON 88302 showed only 1.7% chlorosis at a similar rate at thisstage and no increased chlorosis at double the rate (3600 g ae/ha). Inaddition, MON 88302 event showed no percent growth reduction at the 1800g ae/ha application rate to the 4-6 leaf stage while RT73 had a 5.3%growth reduction. At the 3600 g ae/ha glyphosate rate at the four to sixleaf and first flower crop application stages RT73 showed 10% chlorosis,while MON 88302 showed only 1.7% and 4.7%, respectively. At the 3600 gae/ha glyphosate rate at the four to six leaf and first flower cropapplication stages RT73 showed 20.8% and 8.1% growth reduction,respectively, while MON 88302 showed only 0.3% and 1.1%, respectively.

TABLE 2 Glyphosate Tolerance in RT73 event compared to MON 88302 7-10DAT % 14-21 DAT Glyphosate Rate Crop CHLR % GR Event (g ae/ha) Stage AvgAvg RT73 0 4-6 leaf 0.0 0.0 RT73 450 4-6 leaf 0.3 0.9 RT73 900 4-6 leaf2.2 1.3 RT73 1800 4-6 leaf 4.7 5.3 RT73 3600 4-6 leaf 10.0 20.8 RT73 4501^(st) flower 0.6 0.8 RT73 900 1^(st) flower 1.8 0.3 RT73 1800 1^(st)flower 6.7 3.3 RT73 3600 1^(st) flower 10.0 8.1 MON88302 0 4-6 leaf 0.00.0 MON88302 450 4-6 leaf 0.0 0.3 MON88302 900 4-6 leaf 0.7 0.7 MON883021800 4-6 leaf 1.7 0.0 MON88302 3600 4-6 leaf 1.7 0.3 MON88302 450 1^(st)flower 0.0 0.0 MON88302 900 1^(st) flower 0.3 0.8 MON88302 1800 1^(st)flower 0.9 0.0 MON88302 3600 1^(st) flower 4.7 1.1

Data on the days after planting to swath of canola comprising event RT73compared to canola comprising event MON 88302 are provided in Table 3.Briefly, maturity was delayed significantly in the Genuity™ RoundupReady® Canola system (RT73 event) at the 3600 g ae/ha rate at both thefour to six leaf and first flower applications, likely a consequence ofthe crop injury. No delay in maturity was observed in the MON 88302plants except that there was a significant shortening of maturity at the1800 g ae/ha rate at the four to six leaf application staging.

TABLE 3 Days after Planting to Swath in RT73 event compared to MON 88302Rate g/ha Delta - P Variety Application ae Mean Control Days value RT734-6 leaf 450 103.6 103.8 −0.20 0.45 RT73 4-6 leaf 900 103.6 103.8 −0.250.36 RT73 4-6 leaf 1800 104.3 103.8 0.43 0.12 RT73 4-6 leaf 3600 104.7103.8 0.83 <0.05 RT73 1^(st) flower 450 103.8 103.8 0.00 1.00 RT731^(st) flower 900 104.2 103.8 0.33 0.21 RT73 1^(st) flower 1800 104.3103.8 0.50 0.06 RT73 1^(st) flower 3600 104.7 103.8 0.83 <0.05 MON883024-6 leaf 450 104.7 105.2 −0.46 0.10 MON88302 4-6 leaf 900 105.2 105.20.04 0.90 MON88302 4-6 leaf 1800 104.5 105.2 −0.67 <0.05 MON88302 4-6leaf 3600 105.2 105.2 0.00 1.00 MON88302 1^(st) flower 450 105.1 105.2−0.08 0.75 MON88302 1^(st) flower 900 104.7 105.2 −0.42 0.12 MON883021^(st) flower 1800 104.9 105.2 −0.25 0.34 MON88302 1^(st) flower 3600105.0 105.2 −0.17 0.53

Data on the seed moisture of canola comprising event RT73 compared tocanola comprising event MON 88302 are provided in Table 4. Seed moisturewas measured electronically when the individual plots were harvested.Seed moistures within each system were a comparison between theglyphosate rate sprayed and the unsprayed treatment. Seed moisture inthe Genuity™ Roundup Ready® Canola system was higher at the 1800 g ae/haand 3600 g ae/ha rates at both the four to six leaf and first flowercrop stage applications. The increased seed moisture in these twotreatments again reflects a delay in maturity, which is a result of cropinjury previously described. No significant effects on seed moisturewere observed in the MON 88302 plants.

TABLE 4 Seed Moisture in RT73 event compared to MON 88302 Rate g/haTreated Delta P Variety Application ae Mean Control % value RT73 4-6leaf 450 7.6 7.3 0.4 0.41 RT73 4-6 leaf 900 7.7 7.3 0.4 0.35 RT73 4-6leaf 1800 8.4 7.3 1.1 <0.05 RT73 4-6 leaf 3600 9.8 7.3 2.5 <0.05 RT731^(st) flower 450 7.7 7.3 0.4 0.39 RT73 1^(st) flower 900 8.0 7.3 0.70.09 RT73 1^(st) flower 1800 8.3 7.3 1.0 <0.05 RT73 1^(st) flower 36009.3 7.3 2.0 <0.05 MON88302 4-6 leaf 450 8.0 8.1 −0.1 0.86 MON88302 4-6leaf 900 8.0 8.1 0.0 0.96 MON88302 4-6 leaf 1800 8.0 8.1 −0.1 0.87MON88302 4-6 leaf 3600 7.9 8.1 −0.2 0.69 MON88302 1^(st) flower 450 8.28.1 0.2 0.72 MON88302 1^(st) flower 900 8.3 8.1 0.2 0.60 MON88302 1^(st)flower 1800 8.1 8.1 0.1 0.87 MON88302 1^(st) flower 3600 8.4 8.1 0.30.44

Data on the yield of canola comprising event RT73 compared to canolacomprising event MON 88302 are provided in Table 5 and FIGS. 2 and 3.Yield comparisons were made between the different glyphosate rates andthe unsprayed treatment for canola comprising each event. In theGenuity™ Roundup Ready® Canola system, significant yield reduction wasobserved at the 1800 and 3600 g ae/ha rates at both the four to six leafand first flower applications and the 900 g/ha rate at the first flowerapplication. No significant differences in yield were observed in theMON 88302 plants at any rate or timing.

TABLE 5 Yields in RT73 event compared to MON 88302 Rate g/ha TreatedDelta P Variety Application ae Mean Control bu/ac value RT73 4-6 leaf450 56.0 58.0 −2.0 0.45 RT73 4-6 leaf 900 55.6 58.0 −2.4 0.36 RT73 4-6leaf 1800 51.4 58.0 −6.6 <0.05 RT73 4-6 leaf 3600 40.6 58.0 −17.4 <0.05RT73 1^(st) flower 450 56.1 58.0 −1.9 0.46 RT73 1^(st) flower 900 51.258.0 −6.8 <0.05 RT73 1^(st) flower 1800 48.2 58.0 −9.8 <0.05 RT73 1^(st)flower 3600 41.5 58.0 −16.5 <0.05 MON88302 4-6 leaf 450 53.7 52.2 1.50.57 MON88302 4-6 leaf 900 54.4 52.2 2.2 0.40 MON88302 4-6 leaf 180051.6 52.2 −0.6 0.82 MON88302 4-6 leaf 3600 50.9 52.2 −1.3 0.61 MON883021^(st) flower 450 52.3 52.2 0.1 0.98 MON88302 1^(st) flower 900 50.452.2 −1.7 0.49 MON88302 1^(st) flower 1800 52.5 52.2 0.3 0.91 MON883021^(st) flower 3600 48.4 52.2 −3.8 0.14

The current labeled rate of glyphosate for the Genuity™ Roundup Ready®Canola system is 675 g ae/ha applied once or 450 g ae/ha applied twice.Applications can be made up to the six leaf stage. Rates for theMON88302 may be up to 1800 g ae/ha applied up to the first flower of thecrop. The Genuity™ Roundup Ready® Canola system had 11.4% yieldreduction at the proposed MON 88302 application rate of 1800 g ae/ha and30% yield reduction at 2× the proposed rate (FIG. 2). These yieldreductions were observed at the current labeled Genuity™ Roundup Ready®Canola crop application stage of four to six leaf. No significant yieldreductions were observed in the MON 88302 plants. FIG. 3 shows theyields at the glyphosate rates and crop staging (first flower). Therewas a significant yield reduction in all rates shown for the Genuity™Roundup Ready® Canola system at this later crop staging. No significantyield reductions were observed in the MON 88302 plants.

Example 3 Characterization of MON 88302 DNA Sequences

The DNA inserted into the genome of plant MON 88302 and the genomicsequence flanking the insert was characterized by detailed molecularanalyses. These analyses included: sequencing the insert sequence,determining the insert number (number of integration sites within thegenome), determining the copy number (number of copies of transgene DNAwithin one locus), assessing the integrity of the inserted genecassette, and characterizing the flanking sequences.

Genomic DNA sequences flanking the transgene DNA insertion in event MON88302 were determined using inverse thermal amplification as describedin Ochman et al., 1990 (PCR Protocols: A guide to Methods andApplications, Academic Press, Inc.). Plant genomic DNA was isolated fromnon-transgenic Ebony and different transgenic events arising from theAgrobacterium-mediated transformation of Brassica napus described inExample 1. Tissue used for DNA isolation was produced under standardgreenhouse conditions. Approximately 1 gram of young leaf tissue wascombined with liquid nitrogen and ground to a fine powder using a mortarand pestle. DNA was extracted using a Nucleon™ PhytoPure™ Genomic DNAextraction kit (RPN8511, Amersham, Piscataway, N.J.) according to themanufacturer's protocol. After the final precipitation step, DNA fromindividual samples was resuspended in 0.5 ml of TE (10 mM Tris-HCl pH8.0, 1 mM EDTA). This method can be modified by one skilled in the artto extract DNA from any tissue, including, but not limited to seed.

An aliquot of DNA from each sample was digested with restrictionendonucleases selected based upon restriction analysis of the transgeneDNA. After self-ligation of restriction fragments, thermal amplificationwas performed using primers designed from the transgene DNA sequencethat would amplify sequences using either the ELONGASE® Amplificationsystem (Cat. No. 10481-018, Invitrogen, Carlsbad, Calif.) or the ExpandLong Template PCR System (Cat. No. 1681842, Roche Applied Science,Indianapolis, Ind.) extending away from the 5′ and 3′ ends of thetransgene DNA. Amplicons produced from the reactions were separated byagarose gel electrophoresis and purified using a QIAGEN gel purificationkit (Qiagen, Valencia, Calif.). The gel-purified amplicons were clonedinto the pCR®-XL-TOPO® vector (Invitrogen, Carlsbad, Calif.) followingthe manufacturer's protocol. The resulting plasmids containing the eventMON 88302 flanking genomic sequences were sequenced using standard DNAsequencing protocols. The genomic DNA adjacent to, or flanking, the 5′end of the transgenic DNA inserted into the genome is presented as SEQID NO: 3 ([C], see FIG. 1). The genomic DNA adjacent to the 3′ end ofthe transgenic DNA inserted into the genome is presented as SEQ ID NO: 42([D], see FIG. 1). The segment of the expression cassette DNA that wasfully integrated into the genomic DNA is presented as SEQ ID NO: 5 ([E],see FIG. 1).

Isolated DNA molecule sequences were compared to the transgene DNAsequence to identify the flanking sequence and the co-isolated transgeneDNA fragment. Confirmation of the presence of the expression cassettewas achieved by thermal amplification with primers designed based uponthe deduced flanking sequence data and the known transgene DNA sequence.The wild type sequence corresponding to the same region in which thetransgene DNA was integrated in the transformed line was isolated usingprimers designed from the flanking sequences in event MON 88302. Thethermal amplification reactions were performed using the ELONGASE®Amplification system (Cat. No. 10481-018, Invitrogen, Carlsbad, Calif.).The flanking sequences in event MON 88302 and the Ebony wild typesequence were analyzed against multiple nucleotide and proteindatabases. This information was used to examine the relationship of thetransgene to the plant genome and to evaluate the insertion siteintegrity. The flanking sequence and wild type sequences were used todesign primers for TAQMAN endpoint assays used to identify the events.

Example 4 Event Specific Endpoint TAQMAN® Assays

This example describes an event specific endpoint TAQMAN® thermalamplification method developed to identify event MON 88302 in a sample.Examples of conditions useful with the event MON 88302 Specific EndpointTAQMAN® method are as follows. Step 1: 18 megohm water adjusted forfinal volume of 10 μl. Step 2: 5.0 μl of 2× Universal Master Mix (dNTPs,enzyme, buffer) to a 1× final concentration. Step 3: 0.5 μl Primer-1 andPrimer-2 Mix (resuspended in 18 megohm water to a concentration of 20 μMfor each primer) to 1.0 μM final concentration (for example in amicrocentrifuge tube, the following should be added to achieve 500 μl ata final concentration of 20 μM: 100 μl of Primer SQ20901 (SEQ ID NO: 9)at a concentration of 100 μM; 100 μl of Primer SQ23770 (SEQ ID NO: 10)at a concentration of 100 μM; 300 μl of 18 megohm water). Step 4: 0.2 μlEvent 6-FAM™ MGB Probe PB10164 (SEQ ID NO: 11) to 0.2 μM finalconcentration. Step 5: 0.5 μl Internal Control Primer SQ2563 (SEQ ID NO:17) and Internal Control Primer SQ2564 (SEQ ID NO: 18) Mix to 1.0 μMfinal concentration for each primer. Step 6: 0.2 μl Internal ControlVIC™ Probe PB0751 (SEQ ID NO: 19) to 0.2 μM final concentration. Step 7:3.0 μl Extracted DNA (template) for each sample with one each of thefollowing comprising 1. Leaf Samples to be analyzed; 2. Negative control(non-transgenic DNA); 3. Negative water control (no template); 4.Positive control MON 88302 DNA. Step 8: Thermocycler Conditions asfollows: One Cycle at 50° C. for 2 minutes; One Cycle at 95° C. for 10minutes; Ten Cycles of 95° C. for 15 seconds then 64° C. for 1 minutewith −1° C./cycle; Forty Cycles of 95° C. for 15 seconds then 54° C. 1minute; final cycle of 10° C.

DNA molecules useful in the method are, for example, primers SQ20901(SEQ ID NO: 9) and SQ23770 (SEQ ID NO: 10) and the 6FAM™-labeledoligonucleotide probe PB10164 (SEQ ID NO: 11). Other probes and primersmay be designed based upon the sequences of the transgene insert and/orthe flanking sequences provided herein. SQ20901 (SEQ ID NO: 9) andSQ23770 (SEQ ID NO: 10) when used in these reaction methods with PB10164(SEQ ID NO: 11) produce an amplicon that is diagnostic for event MON88302 DNA. The endpoint TAQMAN amplification method also confirms theintegrity of the template DNA by amplification of FatA, a single-copyendogenous gene within Brassica napus. DNA molecules useful in themethod are, for example, primers SQ2563 (SEQ ID NO: 17) and SQ2564 (SEQID NO: 18) and the VIC™-labeled oligonucleotide probe PB0751 (SEQ ID NO:19). The controls for this analysis include a positive control fromBrassica napus containing event MON 88302 DNA, a negative control fromnon-transgenic Brassica napus, and a negative control that contains notemplate DNA.

The endpoint TAQMAN® thermal amplification method was also used todevelop zygosity assays for event MON 88302. A zygosity assay is usefulfor determining if a plant comprising an event is homozygous for theevent DNA; that is comprising the exogenous DNA in the same location oneach chromosome of a chromosomal pair; or heterozygous for an event DNA,that is comprising the exogenous DNA on only one chromosome of achromosomal pair; or is null for the event DNA, that is wild type. Thisexample describes an event specific endpoint TAQMAN® thermalamplification method developed to determine the zygosity of event MON88302 in a sample. For this assay, a three primer assay was employedwherein primer SQ21948 (SEQ ID NO: 12) hybridizes and extendsspecifically from the inserted exogenous DNA, primer SQ22176 (SEQ ID NO:13) hybridizes and extends specifically from the DNA flanking the 5′side of the inserted exogenous DNA, and primer SQ24635 (SEQ ID NO: 14)hybridizes and extends specifically from the DNA flanking the 3′ side ofthe inserted exogenous DNA. The three primers are diagnostic for theevent. In this example, primer SQ22176 (SEQ ID NO: 13) and primerSQ21948 (SEQ ID NO: 12) and the 6FAM™-labeled oligonucleotide probePB4213 (SEQ ID NO: 15) are diagnostic when there is a copy of theinserted exogenous DNA. In this example, SQ22176 (SEQ ID NO: 13) andprimer SQ24635 (SEQ ID NO: 14) and the VIC™-labeled oligonucleotideprobe PB10787 (SEQ ID NO: 16) are diagnostic when there is no copy ofthe inserted exogenous DNA present in the genomic DNA, i.e. wild-type.When the three primers and two probes are mixed together in a PCRreaction with DNA extracted from a plant homozygous for event MON 88302,there is a fluorescent signal only from the 6FAM™-labeledoligonucleotide probe PB4213 (SEQ ID NO: 15) which is indicative of anddiagnostic a plant homozygous for event MON 88302. When the threeprimers and two probes are mixed together in a PCR reaction with DNAextracted from a plant heterozygous for event MON 88302, there is afluorescent signal from both the 6FAM™-labeled oligonucleotide probePB4213 (SEQ ID NO: 15) and the VIC™-labeled oligonucleotide probePB10787 (SEQ ID NO: 16) which is indicative of and diagnostic a plantheterozygous for event MON 88302. When the three primers and two probesare mixed together in a PCR reaction with DNA extracted from a plantwhich is null for event MON 88302 (i.e. wild type), there is afluorescent signal from only the VIC™-labeled oligonucleotide probePB10787 (SEQ ID NO: 16) which is indicative of and diagnostic a plantnull for event MON 88302, i.e. wildtype. Examples of conditions usefulwith this method are as follows. Step 1: 18 megohm water adjusted forfinal volume of 10 μl. Step 2: 5.0 μl of 2× Universal Master Mix (dNTPs,enzyme, buffer) to a 1× final concentration. Step 3: 0.5 μl of ZygosityPrimers SQ21948, SQ22176, SQ24635 (resuspended in 18 megohm water to aconcentration of 20 uM for each primer) to a final concentration of 1.0μM (for example in a microcentrifuge tube, the following should be addedto achieve 500 μl at a final concentration of 20 uM: 100 μl of Primer 1at a concentration of 100 μM; 100 μl of Primer 2 at a concentration of100 μM; 300 μl of 18 megohm water). Step 4: 0.2 μl Zygosity 6-FAM™ MGBProbe PB4213 (SEQ ID NO: 15) (resuspended in 18 megohm water to aconcentration of 10 μM) to 0.2 μM final concentration. Step 5: 0.5 μlInternal Control Primer SQ22176 (SEQ ID NO: 13) and Internal ControlPrimer SQ24635 (SEQ ID NO: 14) Mix (resuspended in 18 megohm water to aconcentration of 20 uM for each primer) to 1.0 μM final concentrationfor each primer. Step 6: 0.2 μl Internal Control VIC™ Probe PB10787 (SEQID NO: 16) (resuspended in 18 megohm water to a concentration of 10 μM)to 0.2 μM final concentration. Step 7: 3.0 μl Extracted DNA (template)for each sample with one each of the following comprising 1. LeafSamples to be analyzed; 2. Negative control (non-transgenic DNA); 3.Negative water control (no template); 4. Positive control MON 88302 DNA.Step 8: Thermocycler Conditions as follows: One Cycle at 50° C. for 2minutes; One Cycle at 95° C. for 10 minutes; Ten Cycles of 95° C. for 15seconds then 64° C. for 1 minute with −1° C./cycle; Thirty Cycles of 95°C. for 15 seconds then 54° C. 1 minute; final cycle of 10° C. A System9700 or Stratagene Robocycler, MJ Engine DNA Engine PTC-225 thermocyclermay be used. Other methods and apparatus are known to those skilled inthe art that would be useful to produce amplicons for identifying theevent MON 88302 DNA in a biological sample. When conducting thermalamplifications in the Eppendorf Mastercycler Gradient or MJ Engine, thethermocycler should be run in the calculated mode. When conductingthermal amplifications in the Perkin-Elmer 9700, the thermocycler shouldbe set with the ramp speed at maximum.

Example 5 Identification of Event MON 88302 in any MON 88302 BreedingActivity

This example describes how one may identify event MON 88302 within theprogeny of any breeding activity using plants comprising event MON88302. DNA event primer pairs are used to produce an amplicon diagnosticfor event MON 88302. An amplicon diagnostic for event MON 88302comprises at least one junction sequence, provided herein as SEQ ID NO:1 or SEQ ID NO: 2 ([A] and [B], respectively as illustrated in FIG. 1).SEQ ID NO: 1 ([A] of FIG. 1) is a nucleotide sequence corresponding tothe junction of the flanking sequence with the 5′ end of transgeneinsert (positions 762 through 821 of SEQ ID NO: 3 [C], see FIG. 1). SEQID NO: 2 ([B], see FIG. 1) is a nucleotide sequence corresponding to thejunction of the flanking sequence with the 3′ end of transgene insert(positions 313 through 372 of SEQ ID NO: 4 [D], see FIG. 1).

Event primer pairs that will produce a diagnostic amplicon for event MON88302 include primer pairs designed using the flanking sequences (SEQ IDNO: 3 and 4) and the inserted transgenic DNA sequence (SEQ ID NO: 5). Toacquire a diagnostic amplicon in which at least 40 nucleotides of SEQ IDNO: 1 is found, one would design a forward primer molecule based uponSEQ ID NO: 3 from bases 1 through 791 and a reverse primer moleculebased upon the inserted expression cassette DNA sequence, SEQ ID NO: 5from positions 1 through 4427 in which the primer molecules are ofsufficient length of contiguous nucleotides to specifically hybridize toSEQ ID NO: 3 and SEQ ID NO: 5. To acquire a diagnostic amplicon in whichat least 40 nucleotides of SEQ ID NO: 2 is found, one would design aforward primer molecule based upon the inserted expression cassette, SEQID NO: 5 from positions 1 through 4427 and a reverse primer moleculebased upon the 3′ flanking sequence, SEQ ID NO: 4 from bases 343 through1250, in which the primer molecules are of sufficient length ofcontiguous nucleotides to specifically hybridize to SEQ ID NO: 4 and SEQID NO: 5. For practical purposes, one should design primers whichproduce amplicons of a limited size range, for example, between 100 to1000 bases. Smaller (shorter polynucleotide length) sized amplicons ingeneral may be more reliably produced in PCR reactions, allow forshorter cycle times, and be easily separated and visualized on agarosegels or adapted for use in endpoint TAQMAN®-like assays. Smalleramplicons can be produced and detected by methods known in the art ofamplicon detection. In addition, amplicons produced using primer pairscan be cloned into vectors, propagated, isolated and sequenced or can besequenced directly with methods well established in the art. Any primerpair derived from the combination of SEQ ID NO: 3 and SEQ ID NO: 5 orthe combination of SEQ ID NO: 4 and SEQ ID NO: 5 that are useful in aDNA amplification method to produce an amplicon diagnostic for event MON88302 or progeny thereof is an aspect of the present invention. Anysingle isolated DNA polynucleotide primer molecule comprising at least11 contiguous nucleotides of SEQ ID NO: 3, or its complement, that isuseful in a DNA amplification method to produce an amplicon diagnosticfor plants comprising event MON 88302 or progeny thereof is an aspect ofthe present invention. Any single isolated DNA polynucleotide primermolecule comprising at least 11 contiguous nucleotides of SEQ ID NO: 4,or its complement, that is useful in a DNA amplification method toproduce an amplicon diagnostic for plants comprising event MON 88302 orprogeny thereof is an aspect of the present invention. Any singleisolated DNA polynucleotide primer molecule comprising at least 11contiguous nucleotides of SEQ ID NO: 5, or its complement that is usefulin a DNA amplification method to produce an amplicon diagnostic forplants comprising event MON 88302 or progeny thereof is an aspect of thepresent invention.

An example of the amplification conditions for this analysis isdescribed in Example 4 above. However, any modification of these methodsor the use of DNA primers homologous or complementary to SEQ ID NO: 3 orSEQ ID NO: 4 or DNA sequences of the transgene insert (SEQ ID NO: 5) ofevent MON 88302 that produce an amplicon diagnostic for event MON 88302is within the scope of the present disclosure. A diagnostic ampliconcomprises a DNA molecule homologous or complementary to at least onetransgene/genomic junction DNA (SEQ ID NO: 1 or SEQ ID NO: 2 or SEQ IDNO: 7 or SEQ ID NO: 8), or a substantial portion thereof.

An analysis for event MON 88302 in a sample may include a positivecontrol from event MON 88302, a negative control from a comparable plantthat is not event MON 88302 (for example, but not limited to, Brassicanapus), and/or a negative control that contains no genomic DNA. A primerpair that will amplify an endogenous DNA molecule may serve as aninternal control for the DNA amplification conditions. Any fragment of asequence selected from sequences as set forth in SEQ ID NO: 3, SEQ IDNO: 4, or SEQ ID NO: 5 may be used as a DNA amplification primer for theproduction of an amplicon by the methods described in Example 4 and suchan amplicon may be diagnostic for event MON 88302 when using event MON88302 as template for such diagnostic amplification reaction. The use ofthese DNA primer sequences with modifications to the methods describedin Example 4 are within the scope of the invention. The ampliconproduced by at least one DNA primer sequence derived from SEQ ID NO: 3,SEQ ID NO: 4, or SEQ ID NO: 5 that is diagnostic for event MON 88302 isan aspect of the invention.

DNA detection kits, which contain at least one DNA primer of sufficientlength of contiguous nucleotides derived from SEQ ID NO: 3, SEQ ID NO:4, or SEQ ID NO: 5 and that when used in a DNA amplification methodproduces a diagnostic amplicon for a plant comprising event MON 88302 orits progeny, may thus be designed and are an aspect of the invention. Aplant part or seed or commodity product that will produce an amplicondiagnostic for event MON 88302 when tested in a DNA amplification methodis an aspect of the invention. The assay for the event MON 88302amplicon can be performed by using any thermocycler or nucleic acidamplification system that can be used to produce an amplicon diagnosticof event MON 88302 as described herein.

A deposit of a representative sample of event MON 88302 seed disclosedabove and recited in the claims has been made under the Budapest Treatywith the American Type Culture Collection (ATCC), 10801 UniversityBoulevard, Manassas, Va. 20110. The ATCC accession number for thisdeposit is PTA-10955. The deposit will be maintained in the depositoryfor a period of 30 years, or 5 years after the last request, or for theeffective life of the patent, whichever is longer, and will be replacedas necessary during that period.

Having illustrated and described the principles of the presentinvention, it should be apparent to persons skilled in the art that theinvention can be modified in arrangement and detail without departingfrom such principles. We claim all modifications that are within thespirit and scope of the appended claims.

We claim:
 1. A method of producing a Brassica napus plant that toleratesapplication of glyphosate herbicide comprising: a) crossing a glyphosatetolerant Brassica napus plant comprising event MON 88302, arepresentative sample of seed comprising said event having beendeposited under ATCC Accession Number PTA-10955, with a second Brassicanapus plant, thereby producing seed; b) growing said seed to produce aplurality of progeny plants; and c) selecting a progeny plant thatcomprises event MON
 88302. 2. The method of claim 1, wherein saidselecting a progeny plant comprises treating said progeny plants withglyphosate and selecting a progeny plant that is tolerant to glyphosate.3. The method of claim 1, wherein said selecting a progeny plantcomprises identifying a progeny plant comprising a DNA molecule having anucleotide sequence selected from the group consisting of SEQ ID NO: 1,SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 7, and SEQ ID NO:8.
 4. The method of claim 1, wherein said second Brassica napus plantlacks tolerance to glyphosate herbicide.
 5. A method of producing aBrassica napus plant that tolerates application of glyphosate herbicidecomprising: a) selfing a glyphosate tolerant event Brassica napus plantcomprising event MON 88302, a representative sample of seed comprisingsaid event having been deposited under ATCC Accession Number PTA-10955,thereby producing seed; b) growing said seed to produce a plurality ofprogeny plants; and c) selecting a progeny plant that comprises eventMON
 88302. 6. The method of claim 3, wherein said selecting a progenyplant comprises identifying a progeny plant comprising a DNA moleculehaving the nucleotide sequence of SEQ ID NO:
 1. 7. The method of claim3, wherein said selecting a progeny plant comprises identifying aprogeny plant comprising a DNA molecule having the nucleotide sequenceof SEQ ID NO:
 2. 8. The method of claim 3, wherein said selecting aprogeny plant comprises identifying a progeny plant comprising a DNAmolecule having the nucleotide sequence of SEQ ID NO:
 3. 9. The methodof claim 3, wherein said selecting a progeny plant comprises identifyinga progeny plant comprising a DNA molecule having the nucleotide sequenceof SEQ ID NO:
 4. 10. The method of claim 3, wherein said selecting aprogeny plant comprises identifying a progeny plant comprising a DNAmolecule having the nucleotide sequence of SEQ ID NO:
 7. 11. The methodof claim 3, wherein said selecting a progeny plant comprises identifyinga progeny plant comprising a DNA molecule having the nucleotide sequenceof SEQ ID NO:
 8. 12. The method of claim 1, wherein said second Brassicanapus plant comprises tolerance to glyphosate herbicide.
 13. The methodof claim 5, wherein said selecting a progeny plant comprises treatingsaid progeny plants with glyphosate and selecting a progeny plant thatis tolerant to glyphosate.
 14. The method of claim 5, wherein saidselecting a progeny plant comprises identifying a progeny plantcomprising a DNA molecule having a nucleotide sequence selected from thegroup consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO:4, SEQ ID NO: 7, and SEQ ID NO:
 8. 15. The method of claim 5, whereinsaid selecting a progeny plant comprises identifying a progeny plantcomprising a DNA molecule having the nucleotide sequence of SEQ IDNO:
 1. 16. The method of claim 5, wherein said selecting a progeny plantcomprises identifying a progeny plant comprising a DNA molecule havingthe nucleotide sequence of SEQ ID NO:
 2. 17. The method of claim 5,wherein said selecting a progeny plant comprises identifying a progenyplant comprising a DNA molecule having the nucleotide sequence of SEQ IDNO:
 3. 18. The method of claim 5, wherein said selecting a progeny plantcomprises identifying a progeny plant comprising a DNA molecule havingthe nucleotide sequence of SEQ ID NO:
 4. 19. The method of claim 5,wherein said selecting a progeny plant comprises identifying a progenyplant comprising a DNA molecule having the nucleotide sequence of SEQ IDNO:
 7. 20. The method of claim 5, wherein said selecting a progeny plantcomprises identifying a progeny plant comprising a DNA molecule havingthe nucleotide sequence of SEQ ID NO:
 8. 21. The method of claim 3,wherein said second Brassica napus plant comprises tolerance toglyphosate herbicide.